QSpace Community:http://hdl.handle.net/1974/131
Sun, 02 Aug 2015 19:19:45 GMT2015-08-02T19:19:45ZThe Channel Imagehttp://qspace.library.queensu.ca:80/retrieve/244/biochem_image.jpghttp://hdl.handle.net/1974/131
Functional studies of the human 25-hydroxyvitamin D3-24-hydroxylase, CYP24A1http://hdl.handle.net/1974/13470
Title: Functional studies of the human 25-hydroxyvitamin D3-24-hydroxylase, CYP24A1
Authors: Kaufmann, Martin
Abstract: This thesis focuses on the cytochrome P450 CYP24A1, which catabolizes 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) via two pathways commencing with C24-, or C23-hydroxylation and culminating in calcitroic acid or 1α,25-(OH)2D3-26,23-lactone respectively. In my thesis, I set out to identify the molecular determinants of the unique multicatalytic activity of human (h) CYP24A1, using a combination of techniques including homology modeling, site-directed mutagenesis and enzyme activity assays. In total, 42 hCYP24A1 mutants were stably transfected into V79-4 cells, and the metabolism of 1α,25-(OH)2D3 and 9 analogs or prodrugs were studied using HPLC and LC-MS-based techniques. Overall, we observed that mutations at sites in contact with the side-chain of 1α,25-(OH)2D3 above the heme in the homology model, affected the regioselectivity of the protein, and mutations at more distal sites along the substrate cavity affected the access of the substrate or intermediates. Of these mutations, A326G and V391L were of particular interest. Ala326, in the centre of the I-helix in contact with the side chain of 1α,25-(OH)2D3, is a major determinant of the regioselectivity difference between opossum (C23-hydroxylating) and human (C24-hydroxylating) enzymes. When Ala326 was mutated to Gly, as it is in opossum, hCYP24A1 was converted from a C24-hydroxylase, to a C23-hydroxylase, forming 1α,25-(OH)2D3-26,23-lactone. We propose that A326G permits the side chain to enter the substrate binding site more deeply, thus positioning C23 as opposed to C24 above the heme for hydroxylation. Another mutation, V391L in the beta-3a sheet, drastically changed the substrate specificity of CYP24A1 towards the prodrug 1α-hydroxyvitamin D3 (1α-OH-D3), which is not metabolized by the wild-type enzyme. V391L converted hCYP24A1 into a 1α-OH-D3-25-hydroxylase, forming 1α,25-(OH)2D3 which was subsequently catabolized via C24-hydroxylation by the same enzyme. The downstream catabolism of 1α,25-(OH)2D3 formed from 1α-OH-D3 was engineered to proceed via C23-hydroxylation, by introducing a V391L/A326G double mutant. We propose that altered steric contact at C21 is responsible for enabling C25-hydroxylation by V391L. Taken together, we have successfully developed an approach that has identified key structural and functional insights into the mechanism of action of hCYP24A1 that can be potentially applied to study other cytochrome P450 enzymes.
Description: Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-06-07 14:46:40.409Wed, 29 Jul 2015 04:00:00 GMThttp://hdl.handle.net/1974/134702015-07-29T04:00:00ZIdentification of TULP3 as a negative regulator of Hedgehog signalling in the mousehttp://hdl.handle.net/1974/13461
Title: Identification of TULP3 as a negative regulator of Hedgehog signalling in the mouse
Authors: Cameron, Donald
Abstract: The Hedgehog (Hh) family of secreted signalling factors play diverse roles in animal development. In mammals, the Hh ortholog Sonic hedgehog (Shh) is critical for the proper formation of the limbs, central nervous system, and axial skeleton, amoung other tissues. Mutations affecting the function of this pathway during development have severe consequences to the developing embryo and can cause birth defects in humans. Inappropriate activation of the pathway in adult tissues has also been implicated in several human cancers. In recent years several unexpected regulatory factors of the pathway during embryogenesis and in the adult have emerged through genetic studies in the mouse, such as proteins involved in vesicle transport and in the formation and function of primary (non-motile) cilia. Evidence is presented here that the mouse Tubby gene family member Tubby-like protein 3 (Tulp3) plays an important negative regulatory function in the Hh signalling pathway during embryogenesis, a role not previously associated with the Tubby proteins. Embryos lacking Tulp3 develop severe neural tube defects and polydactyly, along with ectopic activation of Shh target genes in the developing limbs and CNS, and altered Shh mediated axon guidance in the developing spinal cord. Moreover, Tulp3 was found to act largely independently of Shh, as compound Tulp3/Shh mutant embryos retain expression of Shh target genes and related abnormalities. Finally, the Tulp3 protein was found to localize to the primary cilium in cultured cells, implicating Tulp3, and possibly other Tubby proteins as regulators of cilium based signalling. These results have important implications in the understanding of the regulation of the Hh pathway, and in the emergence of Hh related birth defects and tumourigenesis.
Description: Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-07-08 13:48:24.258Wed, 29 Jul 2015 04:00:00 GMThttp://hdl.handle.net/1974/134612015-07-29T04:00:00ZQuantitative Investigation into the Mechanisms of Coagulation and Fibrinolysishttp://hdl.handle.net/1974/12723
Title: Quantitative Investigation into the Mechanisms of Coagulation and Fibrinolysis
Authors: Cook, P Michael
Abstract: Coagulation and fibrinolysis are essential processes that ensure a rapid, localized response to vascular damage and subsequent removal once the damage is repaired. These two processes are extensively regulated by various feedback mechanisms and inhibitors. In blood, clotting appears to be an all or none event. Such a phenomenon is governed by thresholding behaviour. Thresholding occurs when a precursor generates a response that can either feedback or be inhibited. Prothrombin activation under conditions in which both of these mechanisms are present was determined to experience thresholding. Thrombin generation initiated by factor Xa in the presence of factor V and antithrombin was fit to a model of prothrombin activation. This model showed that factor Va generation before complete factor Xa inhibition is essential for thrombin generation. Upon factor Va formation, factor Xa becomes highly protected from inhibition, and as a result activates prothrombin more efficiently. These data provided mechanistic detail into the thresholding behavior of coagulation.
Thrombin activatable fibrinolysis inhibitor (TAFIa) suppresses fibrinolysis by removing carboxyl-terminal lysine residues exposed by plasmin (Pn) on fibrin. These cleavages result in reduced plasminogen (Pg) activation and increased Pn inhibition by antiplasmin (AP). The effects of TAFIa on tissue type Pg activator (tPA) inhibition by Pg activator inhibitor type 1 (PAI-1) on fibrin were quantified. High molecular weight fibrin degradation products (HMW-FDPs), a surrogate for Pn-modified fibrin, decreased the rate constant for tPA inhibition 3-fold. In the presence of Pg, HMW-FDPs decreased the inhibition rate constant 30-fold. TAFIa treatment of the HMW-FDPs removed the protection associated with Pg, but not HMW-FDPs. TAFIa also abolished the protection of Pn from AP associated with Pn-modified fibrin in clots formed with Glu-Pg, but did not do so in clots formed with Lys-Pg. These data showed that once Lys-Pg and Lys-Pn are generated, the ability of TAFIa to prolong lysis is greatly reduced. These data identified new mechanisms in which TAFIa can attenuate fibrinolysis.
Description: Thesis (Ph.D, Biochemistry) -- Queen's University, 2015-01-30 10:22:26.148Fri, 30 Jan 2015 05:00:00 GMThttp://hdl.handle.net/1974/127232015-01-30T05:00:00ZCHARACTERIZATION OF TAFI ACTIVATION BY THE THROMBIN-THROMBOMODULIN COMPLEXhttp://hdl.handle.net/1974/12703
Title: CHARACTERIZATION OF TAFI ACTIVATION BY THE THROMBIN-THROMBOMODULIN COMPLEX
Authors: WU, CHENGLIANG
Abstract: Once activated, protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI) initiate anticoagulant and anti-fibrinolytic pathways, respectively. PC and TAFI are activated by the thrombin-thrombomodulin (TM) complex. Endothelial PC receptor (EPCR), which binds PC, enhances the activation of PC, but not TAFI. We investigated PC and TAFI activation on cultured human endothelial cells (HUVEC) in the absence or presence of an antibody against EPCR. In the absence of antibody, PC is the favoured substrate of the thrombin-TM complex. In contrast, in the presence of antibody, PC activation is reduced to a level similar to that of TAFI, suggesting that EPCR acts as a molecular switch that influences substrate preference. PC does not compete with TAFI for activation and vice versa, raising the possibility that the two proteins interact with distinct populations of thrombin-TM complexes on endothelial cell surface.
Based on our previous work, the TM-dependence of TAFI activation by thrombin is mediated through exosite interactions involving a positively-charged patch surrounding Lys 42, Lys 43, and Lys 44 on TAFI. Using TAFI variants with one, two or three of these Lys residues replaced with Ala, we determined that each individual Lys residue contributes equally to overall TAFI activation by thrombin-TM and that they act in a cooperative fashion to promote activation. This cooperative interaction is evident in lysis assays where threshold-like behaviour is evident; thus, the anti-fibrinolytic effect of TAFIa is only attenuated when its generation falls below this threshold.
Capitalizing on this Lys-dependent interaction of TAFI with thrombin-TM, we synthesized 10-amino acid peptide analogs of this region. Analogs with one, two or three Lys residues progressively attenuated TAFI activation by thrombin-TM. This was a specific effect because (a) the peptide analogs had no effect on PC activation, (b) the inhibitory effect was lost if the three Lys residues were replaced with Thr, and (c) epsilon amino-caproic acid, a Lys analog, had no effect. These studies not only suggest that lysine residues at 42/43/44 comprise an exosite that mediates the interaction of TAFI, but not PC, with thrombin-TM, but also identify novel reagents that have potential antithrombotic activity by modulating TAFI activation.
Description: Thesis (Ph.D, Biochemistry) -- Queen's University, 2015-01-20 14:28:04.488Wed, 21 Jan 2015 05:00:00 GMThttp://hdl.handle.net/1974/127032015-01-21T05:00:00Z